Mca1135

The increase in the expression of intercellular adhesion molecule 1 (ICAM-1) on the endothelial cell surface is a well-established marker of endothelial cell activation (31 (link), 32 (link)). Six thousand HMEC-1 cells were grown to confluence in each well of a 96-well plate before exposure to placental EVs in quadruplicate, with and without exogenous VEGF supplementation (100 ng/mL), a Flt-1 neutralizing antibody (10 µg/mL) or both treatments, for 24 h in quadruplicate. Then, cells were washed and cell-based ELISA using a mouse anti-human ICAM-1 antibody (MCA1135, Bio-rad, 1:100) was performed as previously described (32 (link)). o-Phenylenediamine was used as the substrate for detection at 490 nm using an xMark spectrophotometer (Bio-Rad).

Cell surface expression of intercellular adhesion molecule 1 (ICAM-1) is a well-described marker of endothelial cell activation^{13 (link),72 (link)}. Six thousand HMEC-1 cells were grown to confluence before exposure to placental EVs, with and without a TLR-9 antagonist (5 µM, Invivogen, Table 1), for 24 hours in quadruplicate. Then, cells were washed and a mouse anti-human ICAM-1 antibody (MCA1135, Bio-rad, 1:100) was added and incubated at 37 °C for two hours, followed by a goat biotinylated anti-mouse IgG antibody (Jackson ImmunoResearch, 1:2000) and a streptavidin-conjugated HRP (Jackson ImmunoResearch, 1:2000). o-Phenylenediamine, a substrate for HRP, was added at 20 ^oC for detection at 490 nm using an xMark spectrophotometer (Bio-rad).Table 1

Details of nucleotides and primers used.

Name	Sequence (5′ to 3′)
Full 15 kb mtDNA sequence F′	TTA AAA CTC AAA GGA CCT GGC
Full 15 kb mtDNA sequence R′	GTG GGT AGG TTT GTT GGT ATC
NADH dehydrogenase 1 F′	ACG CCA TAA AAC TCT TCA CCA AAG
NADH dehydrogenase 1 R′	GGG TTC ATA GTA GAA GAG CGA TGG
TLR-9 agonist	TCG TCG TTT TCG GCG CGC GCC G (on a phosphorothioate backbone)
TLR-9 antagonist	TTT AGG GTT AGG GTT AGG GTT AGG G (on a phosphorothioate backbone)
β2 microglobulin F′	CAC TGA AAA AGA TGA GTA TGC C
β2 microglobulin R′	AAC ATT CCC TGA CAA TCC C